The overall goal of this application is to develop methods for the investigation of the neurotransmitter and neuromodulator properties of substance P (SP) and its metabolites in vivo. Substance P is an important neuropeptide that has been shown to be involved pain perception, inflammation, and a number of disease states including depression and Alzheimer's. Substance P is released in both the brain and peripheral tissue. It is metabolized by a number of different peptidase enzymes in the brain. Many of its metabolites have also been shown to be neurochemically and/or physiologically active. Recently, we have shown that SP can be transported across the blood-brain barrier (BBB) using an in vitro model of the BBB. Substance P was shown to undergo enzymatic degradation at this barrier to smaller peptides that can also permeate the BBB. In this proposal, methods for monitoring the release, transport, and metabolism of substance P in both central and peripheral tissue will be developed. These methods will then be used to investigate the role of the blood-brain barrier in the distribution of substance P and its metabolites between the brain and peripheral tissue in healthy and diseased animals. In addition, the development of separation-based sensors based on microdialysis and microchip CEEC for measuring the activity of nitric oxide synthase and the production of NO will be explored. Nitric oxide is known to change the permeability of the blood-brain barrier. The effect of substance P on the expression of the inducible form of nitric oxide synthase (INOS) and the release of NO at this barrier will also be investigated.